I would like to thank Mr. Schlenker, Jeff Walsh, Amelia Thomas, and all of the other birds for providing me with the opportunity to take part in an internship at Bluebird Bio this summer. Bluebird Bio is a gene therapy company that works to create one-time cures for patients with rare diseases. They currently have clinical programs for four diseases - sickle cell, dependent beta-thalassemia, cerebral adrenoleukodystrophy, and multiple myeloma - each at different stages of certification. In addition, they are working in preclinical labs to target other diseases.
I had the pleasure of working with Amelia Thomas, a brilliant scientist working with the Vector Biology team. In the ex-vivo gene therapy process, hematopoietic stem cells are taken from the patient's blood to be treated. They are treated with a lentivirus vector, a neutered virus that will only infect a single cell. After being intensely washed and cleaned, the cells are put back into the body. The Vector Biology team works on the lentivirus vector, the envelope, the capsid, and the gene.
As the work I did was preclinical research, it is confidential so please understand the lack of specifics. Amelia is working on an innovative project to change the viral envelope of the plasmid to improve the delivery of the therapy. I cloned the DNA plasmid by cutting the DNA strand in two spots with one restriction enzyme and replacing it with the X envelope through ligation. From here, I put the plasmid into E Coli. and put it onto an agarose plate and allowed it to incubate overnight, creating large quantities of E Coli. colonies. Normally, the control plate should have very few colonies, however, this was not the case when I cloned. My control plate was highly populated with colonies. For this particular X envelope, we needed to use one restriction enzyme. This allowed the plasmid to close without the new envelope, creating colonies on the control plate that do not carry the new envelope. Another outcome of using this type of cloning strategy that can occur in addition to not including the new envelope, is that the envelope can ligate backward. To ensure that everything has ligated correctly, we plucked 12 colonies of each sample and let them incubate and grow. The following day, I performed DNA purification. This process eliminates the unneeded parts of the cells so only the DNA remains. The process took about three hours and consisted of pipetting, centrifuging, vacuuming, and waiting.
To determine if the X envelope had been incorporated into our plasmid, we did a restriction enzyme digest and then analyzed the DNA sample using gel electrophoresis. The gel uses electrical currents to bring the positively charged DNA through and separates the DNA by the size of each stand.
From looking at our plasmid map, we knew that if the DNA had 3 bright bands below the starting point it had incorporated the X envelope. However, if the gel only showed 2 bright bands, the plasmid had bonded without the X envelope. We took note of the correct ones and prepared them for sequencing.
The image on the left shows where the 3 bands should occur in each lane when a plasmid that contains the envelope and was cut by the digest enzyme is present. The other two images are the gel electrophoresis results from my two sets of samples. In the middle image, the green arrow points to a correct lane, as 3 bands line up with the digital image on the left. The red arrow shows a plasmid that did not incorporate the envelope, resulting in only two bands. In the image to the right, the red arrow indicates an incorrect plasmid that was cut into 4 strands.
To sequence the DNA, we took two samples of each correct plasmid and added a primer to each. These primers instruct where the DNA synthesis will start. We use two different primers for each plasmid so we can read the connection of the X envelope at both ends, one primer acts forwards, the other backward. The sequencer sends back a long strand of colored peaks, each color representing a specific base pair.
The top image shows a larger scale sequencing result. The bottom image shows the same image zoomed in.
Once the DNA has been sequenced we can tell if the X envelope has been incorporated with the correct orientation. To do this, we insert the DNA sequence into a mapping program of the correctly constructed plasmid. If the base pairs of the sample match the template, we know the envelope is going the correct way. Furthermore, if the base pairs do not match at the ligation point, the envelope is backward. From here, the correct plasmids move forward to the next stage of the experiment.
My time at Bluebird Bio was invaluable. The culture is laid back and holds true to one of the Bluebird values, “we take what we do seriously, but we don’t take ourselves too seriously.” I believe bluebird bio is truly a special environment that I admire. I am beyond grateful for everything I have learned, the hands-on experience, and the connections and friendships I have made. In addition to learning an immense amount each day, I had fun and enjoyed my time at the “nest.” I hope I will find my way back to bluebird bio in the near future. As bluebirds would say, it was flocking awesome.